New Insight into the Substrate Selectivity of Bovine Milk γ-glutamyl Transferase via Structural and Molecular Dynamics Predictions

Bovine milk γ-glutamyltransferase (BoGGT) can produce γ-glutamyl peptides using L-glutamine as a donor substrate, and the transpeptidase activity is highly dependent on both γ-glutamyl donors and acceptors. To explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, molecular docking and molecular dynamic simulations were performed with L-glutamine and L-γ-glutamyl-p-nitroanilide (γ-GpNA) as donors. Ser450 is a crucial residue for the interactions between BoGGT and donors. BoGGT forms more hydrogen bonds with L-glutamine than γ-GpNA, promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. The BoGGT intermediate forms more hydrogen bonds with Val-Gly than L-methionine and L-leucine, which can promote the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the critical residues responsible for the interactions of donors and acceptors with the BoGGT and provides a new understanding of the substrate selectivity and catalytic mechanism of GGT.


Introduction
Because of its ability to catalyze the transpeptidase reaction to produce γ-glutamyl peptides, γ-glutamyl transferase from bovine milk (BoGGT) has recently received increasing attention [1][2][3]. γ-Glutamyl peptides can be applied as taste enhancers to enhance the mouthfeel and aftertaste of food [4,5]. Furthermore, it has been demonstrated that γ-glutamyl peptides exhibit a variety of biological activities, comprising anti-inflammatory, hypoglycemic, and appetite-suppressing effects [6]. In 2009, γ-glutamyl peptides were identified from Swiss Gruyere and Gouda cheeses [7,8]. Toelstede and Hofmann [7] found an association between the transpeptidase activity and concentration of γ-glutamyl peptides in various cheese types. They demonstrated that BoGGT might produce γ-glutamyl peptides in raw milk cheeses. It has been established that γ-glutamyl peptides in Parmesan cheese are mainly produced by the transpeptidase reaction of BoGGT [3]. Moreover, Yang et al. [9] identified γ-glutamyl peptides from protein hydrolysates of enzyme-modified butter and revealed that BoGGT could produce γ-glutamyl peptides. Our previous study identified BoGGT (G3N2D8) using LC-MS/MS and validated its ability to generate γ-glutamyl peptides from whey protein and casein hydrolysates with L-glutamine as a donor [1,2].
The types of γ-glutamyl donors and acceptors affect the transpeptidase activity of GGT. A recent study documented that Escherichia coli GGT has stereoselectivity for γ-glutamyl acceptors, suggesting that the transpeptidase activity of E. coli GGT depends on the acceptor Our previous study identified BoGGT as the bovine enzyme (G3N2D8) [1]. In the present study, AlphaFold v2.0 was applied to construct the 3D structure of BoGGT. As illustrated in Figure 1A, the majority of the BoGGT residues were predicted with very high confidence, supporting that the 3D structure was accurately predicted.

Interactions between BoGGT and Donor
The catalytic nucleophile of human GGT has already been reported to be Thr381 [15,16]; multiple sequence alignment confirmed that the catalytic nucleophile of BoGGT is Thr380 [1]. It is generally accepted that the hydroxyl oxygen (-OH) on Thr380 from BoGGT will attack the amide carbon of the donor, resulting in the formation of the γ-glutamyl-BoGGT intermediate [20]. Our previous study demonstrated that BoGGT has lower transpeptidase activity with γ-GpNA as a γ-glutamyl donor than the L-glutamine donor [2]. However, the molecular mechanism underlying the difference in donor affinity for BoGGT remains unclear. The per-residue confidence score (pLDDT) is utilized to assign a color to each residue in the model. Residues with extremely high confidence (pLDDT > 90) are shown in blue, while those with high confidence (70 < pLDDT < 90) are shown in cyan. On the contrary, residues with low confidence (50 < pLDDT < 70) are displayed in yellow, while very low confidence residues (pLDDT < 50) are represented in orange. (B) Three-dimensional structure of bovine milk γ-glutamyl transferase with light and heavy chains shown in different colors.

Interactions between BoGGT and Donor
The catalytic nucleophile of human GGT has already been reported to be Thr 381 [15,16]; multiple sequence alignment confirmed that the catalytic nucleophile of BoGGT is Thr 380 [1]. It is generally accepted that the hydroxyl oxygen (-OH) on Thr380 from BoGGT will attack the amide carbon of the donor, resulting in the formation of the γglutamyl-BoGGT intermediate [20]. Our previous study demonstrated that BoGGT has lower transpeptidase activity with γ-GpNA as a γ-glutamyl donor than the L-glutamine donor [2]. However, the molecular mechanism underlying the difference in donor affinity for BoGGT remains unclear. AlphaFold prediction on the three-dimensional structure of bovine milk γ-glutamyl transferase. (A) Three-dimensional structure of bovine milk γ-glutamyl transferase displayed in various colors based on model confidence. Note: The per-residue confidence score (pLDDT) is utilized to assign a color to each residue in the model. Residues with extremely high confidence (pLDDT > 90) are shown in blue, while those with high confidence (70 < pLDDT < 90) are shown in cyan. On the contrary, residues with low confidence (50 < pLDDT < 70) are displayed in yellow, while very low confidence residues (pLDDT < 50) are represented in orange. (B) Three-dimensional structure of bovine milk γ-glutamyl transferase with light and heavy chains shown in different colors.
Molecular docking results showed eight hydrogen bonds between BoGGT and L-glutamine, whereas there were four hydrogen bonds between BoGGT and γ-GpNA. These hydrogen bonds are responsible for glutamine binding at the active site. This might explain why BoGGT has a higher transferase activity with L-glutamine as a γ-glutamyl donor than γ-GpNA. Figure 2A displays a PyMol visualization of the interaction between BoGGT and L-glutamine. The oxygen atom on Gly379 and Thr380 formed a hydrogen bond (H-bond) with the L-glutamine's amide hydrogen atom, respectively. H-bonds were formed between the hydrogen atoms on Ser450, Ser451, and Met452 and the carboxy oxygen atom on L-glutamine. The transpeptidase activity of human GGT was reduced to 1% of the wild-type GGT after site mutagenesis of Ser451 or Ser452 [21], and Ser451 or Ser452 also formed H-bonds with the substrate; this indicates that Ser451 and Ser452 promote the interaction between the active site and substrate. The corresponding residues of Ser451 and Ser452 in human GGT are Ser450 and Ser451 in BoGGT, respectively. Therefore, H-bonds between Ser450, Ser451, and the carboxy oxygen atom on L-glutamine seem to promote the donor substrate binding in the active site, resulting in the formation of the γ-glutamyl-BoGGT intermediate. In addition, the amide hydrogen and carbonyl oxygen atoms on Gly473 formed H-bonds with the L-glutamine's carbonyl oxygen and amide hydrogen atoms, respectively. It has been proposed that the H-bonds between Gly473 and the donor substrate promote the formation of the γ-glutamyl-GGT intermediate [15].
BoGGT and L-glutamine. The oxygen atom on Gly379 and Thr380 formed a hydrogen bond (H-bond) with the L-glutamine's amide hydrogen atom, respectively. H-bonds were formed between the hydrogen atoms on Ser450, Ser451, and Met452 and the carboxy oxygen atom on L-glutamine. The transpeptidase activity of human GGT was reduced to 1% of the wild-type GGT after site mutagenesis of Ser451 or Ser452 [21], and Ser451 or Ser452 also formed H-bonds with the substrate; this indicates that Ser451 and Ser452 promote the interaction between the active site and substrate. The corresponding residues of Ser451 and Ser452 in human GGT are Ser450 and Ser451 in BoGGT, respectively. Therefore, Hbonds between Ser450, Ser451, and the carboxy oxygen atom on L-glutamine seem to promote the donor substrate binding in the active site, resulting in the formation of the γglutamyl-BoGGT intermediate. In addition, the amide hydrogen and carbonyl oxygen atoms on Gly473 formed H-bonds with the L-glutamine's carbonyl oxygen and amide hydrogen atoms, respectively. It has been proposed that the H-bonds between Gly473 and the donor substrate promote the formation of the γ-glutamyl-GGT intermediate [15]. An H-bond occurs between the oxygen atom on Thr398 and the amide hydrogen on L-glutamine. It is universally accepted that the formation of H-bonds between enzyme and substrates greatly affects the specificity of the enzyme and thus facilitates the enzymatic reaction. PyMol visualization of the interaction between BoGGT and γ-GpNA is presented in Figure 2B. The oxygen atom on Gly378 formed an H-bond with the amide hydrogen atom of γ-GpNA. The hydrogen atom on Tyr402, Ser450, and Gly472 formed Hbonds with the oxygen atom on γ-GpNA. Based on the interactions between BoGGT and the γ-glutamyl donor, it could be concluded that Ser450 is a crucial residue for donor substrate recognition.

MD Simulations of BoGGT-Donor Complex
After the molecular docking, the BoGGT-donor complex was employed to conduct 80 ns MD simulations to explore the interaction between BoGGT and the donor substrate. The root-mean-square deviation (RMSD) represents how structures change over time compared to the starting point. The RMSD of the complexes (Cα of BoGGT and the donor substrate) was used to assess the system stability of BoGGT-donor complexes during an An H-bond occurs between the oxygen atom on Thr398 and the amide hydrogen on L-glutamine. It is universally accepted that the formation of H-bonds between enzyme and substrates greatly affects the specificity of the enzyme and thus facilitates the enzymatic reaction. PyMol visualization of the interaction between BoGGT and γ-GpNA is presented in Figure 2B. The oxygen atom on Gly378 formed an H-bond with the amide hydrogen atom of γ-GpNA. The hydrogen atom on Tyr402, Ser450, and Gly472 formed H-bonds with the oxygen atom on γ-GpNA. Based on the interactions between BoGGT and the γ-glutamyl donor, it could be concluded that Ser450 is a crucial residue for donor substrate recognition.

MD Simulations of BoGGT-Donor Complex
After the molecular docking, the BoGGT-donor complex was employed to conduct 80 ns MD simulations to explore the interaction between BoGGT and the donor substrate. The root-mean-square deviation (RMSD) represents how structures change over time compared to the starting point. The RMSD of the complexes (C α of BoGGT and the donor substrate) was used to assess the system stability of BoGGT-donor complexes during an 80 ns simulation. As demonstrated in Figure 3A, both complexes had steady RMSDs after 40 ns simulations, indicating that the L-glutamine and the γ-GpNA donor were tightly bound to BoGGT. The RMSDs of the donor substrates and backbone of BoGGT were calculated to evaluate the stability of the donor substrate and backbone of BoGGT further ( Figure 3B). After a 60 ns simulation, the RMSD of the BoGGT backbone and ligand (L-glutamine and γ-GpNA) from complexes stabilized, indicating that the protein had equilibrated. Nevertheless, the RMSDs of the complexes were notably greater than that of the BoGGT backbone, which might be due to the opening and closing motion of a lid from BoGGT observed during the simulations. The above results indicate that the BoGGTglutamine and BoGGT-GpNA complexes reached a steady state after 60 ns of simulations. For each BoGGT-donor substrate complex, the root-mean-square fluctuations (RMSFs) of its constituent amino acids were determined, as well. Amino acids 121-124 and 431-434 of the BoGGT-Glutamine complexes were found to have higher fluctuation values than those of BoGGT-GpNA, suggesting these residues might be involved in the conformational alteration of the BoGGT-Glutamine complexes ( Figure 3C).  H-bonds between the enzyme and substrate are widely considered to be the primary factor that facilitate the enzymatic reaction [22]. Consequently, H-bond numbers were also utilized to evaluate the binding between the donor substrate and BoGGT. As displayed in Figure 3D, the H-bond numbers between BoGGT and L-glutamine were significantly higher than those between BoGGT and γ-GpNA during the 60-80 ns simulation (p < 0.05), which was consistent with the docking results. This suggests that BoGGT has a higher interaction with L-glutamine than γ-GpNA.
GGT first forms γ-glutamyl-GGT intermediate during the transpeptidase reaction when the hydroxyl oxygen on the active site attacks the carbonyl carbon on the donor substrate [23]. Consequently, during the MD simulations, the distance between the hydroxyl oxygen atom (OG 1 ) on Thr380 and the amide carbon (C) on the γ-glutamyl donor was inspected to evaluate the possibility of the interaction between BoGGT and the donor. As displayed in Figure 3E, the distance between OG 1 on Thr380 and the amide carbon on L-glutamine (0.514 ± 0.031 nm) was significantly (p < 0.05) lower than that for BoGGT and γ-GpNA (0.749 ± 0.042 nm) during the 60-80 ns simulations.
Previous research has reported that Pro427-Ser438 of human GGT form a lid-loop, which may affect the substrate binding in the active site [24][25][26]. Sequence alignment showed that the lid-loop of BoGGT is Pro426-Ser437, and the opening and closing motion was observed during the MD simulation of BoGGT-donor complexes, which could recognize the donor substrate and regulate the access of the donor to the active site. γ-GpNA is a bigger molecule than L-glutamine, allowing L-glutamine to access the active site easily. This could also explain why BoGGT has higher transpeptidase activity with L-glutamine as a donor than γ-GpNA. The H-bond numbers between L-glutamine and BoGGT were significantly (p < 0.05) greater than those of γ-GpNA, which can decrease the OG 1 -C distance and thus promote the binding affinity between BoGGT and L-glutamine.

Construction and Optimization of the BoGGT Intermediate
Our previous study revealed that the transpeptidase activity of BoGGT is highly dependent on the acceptor with both L-glutamine and γ-GpNA donors [2]. In the second step of the transpeptidation reaction, it is well established that the γ-glutamyl-GGT intermediate reacts with acceptors to produce γ-glutamyl peptides. To explore the interaction mechanism between the γ-glutamyl-BoGGT intermediate and acceptors, the γ-glutamyl-BoGGT intermediate was constructed and optimized using GROMACS. After constructing the γ-glutamyl-BoGGT intermediate, the intermediate was optimized using 100 ns MD simulations. Figure 4B presents the γ-glutamyl-BoGGT intermediate, which is the combination of the hydroxyl oxygen on Thr380 of BoGGT with the γ-glutamyl group from the donor. As demonstrated in Figure 4C

Interactions between the BoGGT Intermediate and Acceptors
Our previous study demonstrated that the transpeptidase activity of BoGGT with L-methionine as an acceptor substrate was significantly lower than that of Val-Gly, whereas it was significantly higher than that of L-leucine [2]. Hillmann, Behr, Ehrmann, Vogel and Hofmann [3] also proposed that L-methionine was a better acceptor substrate than L-leucine for BoGGT. However, the molecular mechanism behind the difference in acceptor affinity for the BoGGT intermediate remains unclear. In the current study, molecular docking was utilized to explore the interactions between the BoGGT intermediate and γ-glutamyl acceptors.
As shown in Figure 5A, molecular docking of the BoGGT intermediate and Val-Gly showed five hydrogen bonds; three residues (Gly379, Ile399, and Asn400) were involved in the formation of H-bonds. The oxygen atom on Gly379 formed an H-bond with the hydroxyl hydrogen atom on Val-Gly. Two H-bonds were formed between the hydrogen atoms on Ile399 and Asn400, and the carboxyl oxygen atom on Val-Gly, and one H-bond was formed between the hydrogen atom on Asn400 and the hydroxyl oxygen atom on Val-Gly. Four H-bonds were formed between four residues (Ser81, Gly379, Ile399, and Asn400) from the intermediate and L-methionine ( Figure 5B), whereas there were three H-bonds between Gly379, Ile399, and Asn400 on the intermediate and L-leucine ( Figure 5C).
The hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-methionine, respectively. The oxygen atom on Ser81 formed an H-bond with the amide hydrogen atom on L-methionine, and carboxyl oxygen on Gly379 formed an H-bond with the hydroxyl hydrogen atom on L-methionine. Regarding the interaction between the intermediate and L-leucine, the hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-leucine, respectively. The carboxyl oxygen on Gly379 formed an H-bond with the amide hydrogen atom on L-leucine. It could be concluded that Gly379, Ile399, and Asn400 are key residues responsible for the interactions between the intermediate and acceptors, thereby regulating the transfer of the γ-glutamyl moiety from the γ-glutamyl-BoGGT intermediate to the acceptor.

Interactions between the BoGGT Intermediate and Acceptors
Our previous study demonstrated that the transpeptidase activity of BoGGT with Lmethionine as an acceptor substrate was significantly lower than that of Val-Gly, whereas it was significantly higher than that of L-leucine [2]. Hillmann, Behr, Ehrmann, Vogel and Hofmann [3] also proposed that L-methionine was a better acceptor substrate than L-leucine for BoGGT. However, the molecular mechanism behind the difference in acceptor affinity for the BoGGT intermediate remains unclear. In the current study, molecular docking was utilized to explore the interactions between the BoGGT intermediate and γglutamyl acceptors.
As shown in Figure 5A, molecular docking of the BoGGT intermediate and Val-Gly showed five hydrogen bonds; three residues (Gly379, Ile399, and Asn400) were involved in the formation of H-bonds. The oxygen atom on Gly379 formed an H-bond with the hydroxyl hydrogen atom on Val-Gly. Two H-bonds were formed between the hydrogen atoms on Ile399 and Asn400, and the carboxyl oxygen atom on Val-Gly, and one H-bond was formed between the hydrogen atom on Asn400 and the hydroxyl oxygen atom on Val-Gly. Four H-bonds were formed between four residues (Ser81, Gly379, Ile399, and The hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-methionine, respectively. The oxygen atom on Ser81 formed an H-bond with the amide hydrogen atom on L-methionine, and carboxyl oxygen on Gly379 formed an H-bond with the hydroxyl hydrogen atom on L-methionine. Regarding the interaction between the intermediate and L-leucine, the hydrogen atom on Ile399 and Asn400 formed one H-bond with the same hydroxyl oxygen atom on L-leucine, respectively. The carboxyl oxygen on Gly379 formed an H-bond with the amide hydrogen atom on L-leucine. It could be concluded that Gly379, Ile399, and Asn400 are key residues responsible for the interactions between the intermediate and acceptors, thereby regulating

MD Simulations of BoGGT Intermediate-Acceptor Complex
After the molecular docking, the BoGGT intermediate-acceptor complexes were used to perform 40 ns MD simulations to explore the interaction between the intermediate and acceptors. As shown in Figure 6A In the second step, the nitrogen atom on the acceptor will attack the oxygen atom on GGT380 (γ-glutamyl-threonine) to produce γ-glutamyl peptides. Therefore, the distance between the oxygen atom on residue 380 and the nitrogen atom on the acceptor is very important to produce γ-glutamyl peptides. As presented in Figure 6B, during the 30-40 ns simulations, the distance between the oxygen atom (O) on GGT380 and the nitrogen atom on Val-Gly (N) (0.814 ± 0.022 nm) was significantly (p < 0.05) lower than that of L-methionine (0.917 ± 0.039 nm) and L-leucine (3.244 ± 0.094 nm), indicating that the intermediate has a higher interaction possibility with Val-Gly. It is generally accepted that H-bonds between enzyme and substrate are the main driving force for the enzymatic reaction. Consequently, the number of H-bonds was also employed to evaluate the interactions between the intermediate and acceptors. As shown in Figure 6C, during the 30-40 ns simulations, the number of H-bonds between the BoGGT intermediate and Val-Gly were significantly higher than that of L-methionine and L-leucine (p < 0.05), which could decrease the O-N distance and thus promote the transfer of the γ-glutamyl moiety from the BoGGT intermediate to Val-Gly. To further validate the binding affinity between the acceptor substrate and the BoGGT intermediate, MMPBSA analysis was employed to estimate the binding affinity between the complexes during MD simulations. As shown in Table 1

Structure Construction of BoGGT and Molecular Docking between γ-glutamyl Donor and BoGGT
In the current study, the three-dimensional (3D) structure of bovine GGT (UniProt no. G3N2D8) was produced by AlphaFold (v2.0) on an in-house system equipped with an A40 GPU running Docker [19,27]. Multiple sequence alignment was performed against the reduced database (db_preset = reduced_dbs), max_template_date set to the enzymes and model_preset as monomer. Each of the five relaxed structures was manually inspected for defects, and the structure with the highest score was selected for further analysis.
The 2D structures of the donor substrates (L-glutamine or L-γ-glutamyl-p-nitroanilide) were obtained from PubChem, and the 3D structures were converted using ChemSketch (ACD Labs, Toronto, ON, Canada). This allowed us to generate models for donor substrate-enzyme complexes by molecular docking using Autodock Vina with default parameters [28,29]. The docking results of ligand-enzyme complexes were ranked based on the binding energy, and the best match was chosen to analyze the interactions between BoGGT and the donors in PyMOL (Schrödinger Inc., New York, NY, USA).

MD Simulation of BoGGT-Donor Complex
GROMACS (version 2022.2) was utilized to conduct MD simulations for BoGGT-Gln and BoGGT-GpNA complexes, based on our previous study [30]. Force field parameters of BoGGT were written in Charmm36 all-atom force field format (July 2021), and force field parameters of L-glutamine and γ-GpNA were generated by the CHARMM General Force Field (CGenFF, https://cgenff.umaryland.edu, accessed on 6 September 2022). In the first stage, a steep descent approach of 50,000 steps was adopted to minimize energy and achieve a stable conformation. After minimizing the system's energy, we performed an isochoric-isothermal equilibration (300 K) for 100 ps, followed by an isothermal-isobaric equilibration (300 K, 1.0 bar) for 100 ps. The complexes were subsequently subjected to 80 ns MD simulations, meaning that the simulations were run in blocks of 80 ns until RMSD simulations were stable within this time course.
The RMSD, RMSF, and H-bond number between BoGGT and donors (L-glutamine and γ-GpNA) were calculated after the MD simulations. To evaluate their interactions, the distance between the amide carbon (C) on the donor and hydroxyl oxygen (OG 1 ) on the active site was also calculated.

Construction of the BoGGT Intermediate and Optimization of the Intermediate
GGT first interacts with a γ-glutamyl group to form γ-glutamyl-GGT intermediate, which then combines with acceptors to synthesize γ-glutamyl peptides [15]. To reveal the mechanism between the γ-glutamyl-BoGGT intermediate and acceptors, the γ-glutamyl-BoGGT intermediate was constructed and optimized using GROMACS. The γ-glutamyl-BoGGT intermediate was formed when hydroxyl oxygen on the active site of BoGGT (Thr380) attacked the carbonyl carbon of the γ-glutamyl group from the donor. In other words, the γ-glutamyl-BoGGT intermediate is the combination of the hydroxyl oxygen of Thr380 on BoGGT with the γ-glutamyl group from the donor. The γ-glutamyl-threonine is not a standard residue in GROMACS; we need to define it in the existing force field for GROMACS to recognize this residue as an amino acid. Briefly, the atoms and linkage information for the new residue was added to the RTP file (aminoacids.RTP), and then the information about how to add hydrogens to the new residue was supplemented to the HDB file (aminoacids.HDB). Afterward, the information about modifying the residue if it is a terminal residue was added to the r2b file. The new residue was supplemented to residuetypes.dat with the name of GGT into the working directory. The γ-glutamyl-threonine residue was built and aligned with the Thr380 of BoGGT, and then the original Thr380 was deleted to obtain the γ-glutamyl-BoGGT intermediate. Subsequently, a 100 ns MD simulation (300 K, 1.0 bar) was applied to optimize the γ-glutamyl-BoGGT intermediate.

Molecular Docking between the BoGGT Intermediate and γ-glutamyl Acceptors
Molecular docking between the BoGGT intermediate and acceptors was performed using Autodock Vina [28,29]. Briefly, the 2D structures of acceptor substrates (Val-Gly, L-methionine, and L-leucine) were obtained from PubChem, and the 3D structures were converted using ChemSketch (ACD Labs, Toronto, ON, Canada). This allowed us to generate models for acceptor substrate-enzyme complexes by molecular docking using Autodock Vina with default parameters [28,29]. The docking results of ligand-enzyme complexes were ranked based on the binding energy, and the best match was chosen to analyze the interactions between the BoGGT intermediate and acceptors in PyMOL (Schrödinger Inc., New York, NY, USA).

MD Simulation of BoGGT Intermediate-Acceptor Complexes
GROMACS was utilized to conduct MD simulations for BoGGT intermediate-acceptor complexes, based on our previous study [30]. Force field parameters of the BoGGT intermediate were written in Charmm36 all-atom force field format (July 2021), and force field parameters of acceptor substrates were generated by the CHARMM General Force Field (CGenFF, https://cgenff.umaryland.edu, accessed on 15 November 2022). In the first stage, a steep descent approach of 50,000 steps was adopted to minimize energy and achieve a stable conformation. After minimizing the system's energy, we performed an isochoric-isothermal equilibration (300 K) for 100 ps, followed by an isothermal-isobaric equilibration (300 K, 1.0 bar) for 100 ps. The complexes were subsequently subjected to MD simulations that were run in 40 ns blocks until a time course of relatively stable RMSD was observed.
RMSDs and H-bond numbers between the intermediate and acceptors were calculated after the MD simulations. To evaluate their interactions, the distance between the amide nitrogen (N) on the acceptor and hydroxyl oxygen on the active site was also calculated. The molecular mechanics Poisson Boltzmann surface area (MMPBSA) was applied to determine the binding free energy using the g_mmpbsa package [31]. Binding energy was calculated using the final 10 ns from the 40 ns MD simulations. Bonded and non-bonded interactions in the vacuum were utilized to estimate the binding affinity of the BoGGT intermediate with acceptors in the solvent stage. The Poisson Boltzmann equation and Solvent Accessible Surface Area (SASA) were utilized to determine the polar and non-polar solvation energy.

Statistical Analysis
The MD simulations were performed in triplicate. The obtained data are presented as mean ± standard deviation. To test differences among means of multiple groups, one-way analysis of variance (ANOVAs) with Duncan's analysis was utilized. To determine whether the difference in means between the two groups was statistically significant, a t-test was used. A p-value less than 0.05 (p < 0.05) indicates that there are significant differences between the samples. The figures and ANOVAs were created using GraphPad Prism 9.3.1 and SAS 9.4, respectively.

Conclusions
BoGGT is an enzyme that could produce γ-glutamyl peptides with L-glutamine as a donor substrate and has the potential as a catalyst to produce taste enhancers, whereas the transpeptidase activity is highly dependent on the γ-glutamyl donors and acceptors. In summary, to explore the molecular mechanism behind the donor and acceptor substrate preferences for BoGGT, structure prediction of BoGGT, molecular docking, and molecular dynamic simulations were performed. The light subunit of BoGGT (Thr380, Thr398, Ser450, Ser451, Met452, and Gly473) is responsible for forming the majority of the H-bonds with the L-glutamine donor. Ser450 is a crucial residue for both L-glutamine-BoGGT and GpNA-BoGGT interactions. H-bond numbers between L-glutamine and BoGGT were significantly higher than that of γ-GpNA, which can decrease the distance between the hydroxyl oxygen atom on the active site of BoGGT and the amide carbon on the donor substrate (OG 1 -C), thereby promoting the binding affinity between BoGGT and L-glutamine. Gly379, Ile399, and Asn400 are crucial residues for the interactions between the BoGGT intermediate and acceptors. H-bond numbers between the BoGGT intermediate and Val-Gly were significantly higher than that of L-methionine and L-leucine, which can decrease the O 1 -N distance, thereby promoting the transfer of the γ-glutamyl group from the intermediate to Val-Gly. This study reveals the interactions between BoGGT and the donor, as well as the interactions between the intermediate and the acceptor substrate, and provides a meaningful method for selecting donors and acceptors with higher affinity for GGT, which can extend the understanding of the catalytic mechanism and promote the yields of γ-glutamyl peptides.